Recovery of enzymes from pancreas gland residues subsequent to insulin removal



Aug. 7, 1956 L. C. MAXWELL ETAL RECOVERY OF ENZYMES FROM PANCREAS GLAND RESIDUES SUBSEQUENT TO INSULIN REMOVAL Filed NOV. 30, 1951 PANCREAS RES/DUE t AC/D/F/ED WATER CONDENSER STORAGE TANK r cow WATER RECOVERED ALCOHOL m/vK STEAM STILL ze $754M I i r-' H :\"1 i F i i l t CEN TRIIFUGE 1 HEAVY FAT I l A F P-A A I I & "/4 I -/T: C D 1 J I BR/NE I C) 1 ,7

I SPENT I RES/DUE EXTRACTOR 1 I l FAT Km J L J SK/MME/P ExmAcr TANK g INVENTORS: i0 45 wit l0 respectively of chymo-trypsin and trypsin.

RECOVERY OF ENZYMES FROM PANCREAS GLAND RESIDUES SUBSEQUENT T INSULIN REMOVAL Loyal C. Maxwell and William M. Thompson, Chicago, Ill., assignors to Armour and Company, Chicago, 111., a corporation of Illinois Application November 30, 1951, Serial No. 259,091

6 Claims. (Cl. 195-66) This invention relates to the recovery of proteolytic enzymes from pancreas gland residues subsequent to insulin removal.

It has long been known that a number of proteolytic enzymes are obtainable from the pancreas glands of mammals. Commercially, these enzymes are mainly obtained from .the pancreas glands of cattle, hogs, and

and ribonuclease appear promising.

Then enzymes are present in the pancreas glands in the form of pro-enzymes or zymogens, which are the precursors of the active enzymes, for example as chymotrypsinogen and trypsinogen, which are the precursors Activation of the enzymes is undesirable, if the insulin is to be extracted from the glands, since when activated they bring about the destruction of the insulin. For this reason, insulin is extracted from the glands with a solvent which effectivelyv keeps the enzymes in an inactive state. Various acidified, water-miscible organic solvents for insulin can be used for this purpose, such as the lower alcohols and ketones. anol, and acetone, and in the United States because of its availability ethanol is employed almost exclusively. The ethanol or similar organic solvent is diluted with water and acidified to prepare the extracting solvent. In order to keep the enzymes inactive and insolubilized, it is preferable to have the extracting solvent contain from about 50 to 85% organic solvent, and to be at a pH below 4.

, To prevent any possibility of confusion, it is desired to point out that the terms enzymes and proteolytic enzymes are used in the following specification and claims to refer to both the pro-enzymes and to the active enzymes derived therefrom.. However, wherever the inactive and active states of the enzymes are of importance more specific terminology is employed.

For many years the pancreas, gland residues resulting from the extraction of insulin from pancreas glands were discarded. No attempt was made to recover the enzymes from these residues, and instead fresh pancreas glands were extracted to obtain the enzymes. due to. the general belief that the enzymes were destroyed by the high concentrations of organic solvents used in the insulin extraction. However, it is now known that .the enzymes are not destroyed but only precipitated or denatured; and that they can be converted back to their normal state by reducing the concentration of the organic solvent to a sufliciently low value, that is, by reducing the volumetric ratio of solvent to water. With the commonly used organic solvents (ethanol, methanol, and

This was United States Patent 0 The preferred solvents are methanol, eth- 'acetone), it has been believed to be suflicient to reduce vof the equipment for carrying out the process.

2,758,055 Patented Aug. 7, 1956 "Ice 2 the concentration of the organic solvent to below 20% by volume.

Raw pancreas glands contain approximately water, and therefore this water must be allowed for in extracting the glands with the proper concentration of organic solvent. The insulin, of course, is distributed in the solid portions of the glands. To facilitate the extraction of the insulin it is customary to comrninute or hash the glands to particles about one-fourth to onehalf in diameter, and then to contact the comminuted gland tissue with the extracting solvent. As indicated above, to keep the enzymes inactive it is necessary that the extracting solvent contain from about 50 to 85% by volume of the organic solvent, which is generally ethanol. It is generally agreed that the preferred concentration of ethanol in the aqueous extracting solvent is about 60 to 75%. The water-organic solvent mixture is generally acidified with sulphuric or hydrochloric acid. Phosphoric acid can also be used, and in fact has a number ofadvantages over other acids. The use of phosphoric acid in extracting insulin from pancreas glands is described in detail in co-pending application United States Serial No. 158,928, filed April 28, 1950, now Patent No. 2,595,278. After the contacting of the solvent with the gland material for a sufiicient period to extract the insulin, the supernatant is separated from the residue, generally by centrifugation. The residue thus obtained generally contains from about 30 to 40% solids, or in other words 60 to 70% of the residue is the extracting solvent containing the high concentration of organic solvent. It may contain as little as 50% liquid.

Heretofore the method of reducing the concentration of the organic solvent in the residue to below 20% has been the dilution of the organic solvent by adding water to the pancreas residues. To produce the required degree of dilution, it has been necessary to add from about 7 to 10 parts by weight of water to each part by weight of residue, depending on the concentration of organic solvent in the extracting solvent. The solids of the residue are then kept in contact with the water containing less than 20% of the organic solvent for at least from 24 to 48 hours so that the water can extract the enzymes. To prevent destruction of the enzymes during the extracting period, it has been customary to keep the extracting solvent at a low temperature (0 to 10 C.). This method of extracting the enzymes is unsatisfactory in a number of Ways. One of its disadvantages is that the dilution method of reducing the concentration of the organic solvent produces excessive volumes of material which are difiicult to handle and increase the cost Another disadvantage is that a long extraction time is required, which greatly decreases the amount of product that can ,be produced in a given period of time with the available equipment. In fact, even when a 48 hour extraction period is employed, the extraction of the enzymes is far from complete. Depending somewhat on the character of the particular pancreas tissue, the amount of agitation, and other factors, it is only possible to extract from 6010 of the enzymes in a 48 hour period by conventional procedures. These problems suggest the search for alternatives, but thus far no satisfactory alternative procedures have been developed. It'has been suggested that the pancreas residues could be pressed to separate the liquid from the solid portions thereof, but this has proved to be impractical for large scale commercial operations. Also, flash heating of the solvent during the extracting period has been tried. In order to prevent destruction of the enzymes, it has not been possible to prolong the flash heat beyond a few minutes, and therefore ithas not proven to be of much value in accelerating the rate of extraction.

It is therefore an object of this invention to develop a process for recovering proteolytic enzymes from the solvent-rich residues resulting from the extraction of insulin from comminuted pancreas glands by contacting the glands with an organic solvent for the insulin, which process will substantially overcome the difiiculties with the prior process described above. More specifically, it is an object of this invention to provide a method for recovering the enzymes from the pancreas residues which will greatly reduce the volumes of material which it is required to handle during the extraction period and thereafter. It is a further object of this invention to shorten the required period of extraction without reducing the completeness of the extraction of the enzymes. Further objects and advantages will appear as the specification proceeds.

This invention is based on the discovery that the above objects can be substantially achieved by removing the organic solvent by distilling the pancreas residues under reduced pressure. The preferred method involves adding a limited number of volumes of water to the pancreas gland residues subsequent to insulin extraction, adjusting the pH of the mixture to a critically low value, and distilling off the organic solvent under reduced pressure.

By the procedure of this invention a more complete extraction of the enzymes can be obtained by adding as little as 3 parts by weight of water to each part by weight of total gland residues, as by the previous method when 7 to 10 parts of water were added. On the basis of the solids in the residues, the amount of water to be added to the residues can vary from none to about parts by weight to each part by weight of residue solids, depending on the ratio of liquid to solids in the residues and on the concentration of ethanol or other organic solvent in the liquid portion. However, it is highly advantageous to add all of the water required for the extraction before the distillation step. Preferably, 6 to 14 parts of water are added to each part of solids. For example, when the liquid portion is 70% of the residue and contains from 60 to 75% organic solvent, excellent results are obtained by adding 10 parts of water to each part of residue solids.

On the basis of the prior knowledge and practice in the field, it would not have been thought possible to remove the alcohol by distillation because it would have been thought that the heating of the solvent and residues therein to the required temperatures would destroy a substantial portion of the enzymes. However, during the experimental work leading to this invention it was discovered that by having the pH of the mixture on the acid side (below pH 6.5) that the destruction of the enzymes could be controlled for a long enough period of time at the temperatures required for vacuum distillation to allow the organic solvent to be removed by this method. It has been determined that the pH of the mixture should be below pH 4 if enzyme activation and resulting destruction is to be held at a minimum. Optimum results appear to be obtained at between pH 1.5 to pH 2.5.

Since the liquid portion of the residues will already contain a suificient quantity of an acid (such as hydrochloric, sulfuric and phosphoric) to be at a pH of 2 to 4, it is not essential that additional acid be incorporated in the slurry charged to the distillation zone to maintain the pH thereof within the stated range during distillation. Even when a considerable volume of water is added to the residues, as is preferred, the resulting pH value may be below pH 4, and possibly even as low as pH 2.5. However, to adjust the pH to the optimum range it will usually be necessary to add an acid to the slurry.

In general, the strong inorganic acids, such as sulphuric or hydrochloric acids, are the preferred reagents for adjusting the pH of the mixture prior to the distillation step. While hydrochloric acid can be used successfully to prevent the destruction of the enzymes, it has certain collateral disadvantages which make it less desirable than sulphuric acid. One of these, of course, is that sulphuric acid is not as corrosive to stainless steel, or alternatively does not require the use of glass-lined equipment. The main disadvantage of hydrochloric acid, however, as compared with sulphuric acid, is that it complicates the filtration of the extract at a later point in the process. The preferred means of adding the acidic reagent is to first disperse it in the water, and then to add the water to the residue. The addition of concentrated sulphuric or hydrochloric acid to the residues may produce a local destruction of the tissue, and also increase the difficulty of producing a uniform mixture.

The residues obtained from the insulin extraction process are relatively difiicult to transport from one part of the plant to another. By immediately adding the water for the extraction of the enzymes to the residues, it is then much easier to pump the mixture to any de sired place for the subsequent process steps. Also, the pumping of the mixture into the equipment for the distillation step assists in mixing the acidified water with the pancreas residue.

Various types of equipment can be employed for carrying out the distillation step. For example, in batch operation a steam-heated pot-type vacuum still can be satisfactorily employed. The removal of the ethanol or other organic solvent by distillation accomplishes two results at the same time. The first result, of course, is the reduction of the concentration of the organic solvent for the purpose of converting the precipitated enzymes back to their original state so that they can be extracted from the residues. The second result is that a considerable amount of the enzymes are extracted from the residues during the distillation, which substantially shortens a subsequent extraction step. In other words, because of the moderately elevated temperature of the mixture during the distillation step and the continual agitation and cavitation of the mixture, the pro-enzymes can be largely extracted in one or two hours without destroying any substantial amount of the pro-enzymes.

As indicated above, it was formerly thought that it was sufficient to reduce the concentration of the ethanol or other organic solvent in the liquid portion of the residue to less than 20% to convert the precipitated enzymes back to their original condition. However, it has now been discovered that the extraction of the enzymes is facilitated and that a more complete extraction can be obtained by reducing the concentration of the organic solvent to a much lower value than 20%. This may possibly be due to the fact that each enzyme appears to be a complex of closely related chemical compounds of varying solubilities. This seems to be especially true of trypsin, which at present is one of the most commercially valuable enzymes, and therefore it is particularly desired to increase the yield of trypsin. Greatly improved yields of the enzymes, and particularly of trypsin, can be obtained by reducing the concentration of the organic solvent to below 4%, and optimum results are obtained at around 1% or less. In other words, it is desired to effect a substantially complete removal of the organic solvent from the mixture.

It has been previously pointed out that the enzymes are subject to destruction by heat, but that when the pancreas residues are in contact with a solvent having a pH at least below 6.5 and preferably not over 4, that the destruction of the enzymes can be prevented for a sufiicient period of time to carry out the removal of the organic solvent by distillation. chymotrypsin is especially subject to destruction by heat. If it is desired to recover the chymotrypsin together with the other enzymes it is necessary to keep the temperature of the mixture below R, if this temperature is to be maintained for the time required to remove the organic solvent by distillation. Other of the enzymes also begin to become subject to excessive destruction at temperatures above 100 F.

was

Hewever','when employing sustained heating of the mixture for commercial production of the enzymes, about 85 F. is the maximum temperature that can be satisfactorily employed. The optimum temperature for commercial operation is around 70 F. Lower temperatures can be employed depending on the degree of vacuum that can be maintained in the distillation zone. With the best high vacuum equipment temperatures as low as 60 F. probably can be employed. However, at temperatures between 70 to 85 F. it is possible to use ordinary vacuum-producing equipment, since in this range a vacuum of 28 to 30 inches'of mercury "is sufficient to carry out the distillation.

The time required for the distillation will of course vary with the degree of vacuum and the temperature of the mixture. The upper limit on the time is determined by the point at which the destruction of the enzymes begins to become excessive. For example, the destruction of trypsin will become excessive after the mixture is heated for hours at 100 F;, and most of the other enzymes will be seriously attacked at lower temperatures and shorter times. The longer the time of the distillation step, the more complete will be the removal of the organic solvent and also the more complete will be the extraction of the enzymes. For commercial operation, it is preferred to heat the mixture at a temperature below about 85 F. for a period of less than 8 hours. Best results appear to be obtained by heating the mixture for from 4 to 6 hours at temperatures of between about 70 to 85 F. during the distillation.

Subsequent to the distillation step, the residue can be separated from the supernatant, and the enzymes recovered from the supernatant. If desired, the extraction of the enzymes from the residue can be continued by other means to obtain a complete extraction of the enzymes. In the co -pending application United Statbs Serial No. 259,090 filedNovember 30, 1951, now U. S. Patent No. 2,686,148, a method of treating the residues from the distillation step to produce a complete extraction of the enzymes therefrom is described in detail. However, since 75 to 80% of the enzymes are extracted during the distillation step, it will be apparent that the process can be satisfactorily carried out by merely separating the residue from the supernatant after the distillation step, and recovering the enzymes from the liquid. The residue can be separated from the extract by any suitable means such as centrifugation and filtration.

The supernatant will then contain the enzymes in the form of their precursors which are termed the proenzymes, as explained above. The pro-enzymes can be separated and activated by well-known procedures. The various enzymes and inert protein impurities have dif' ferential solubilities in aqueous ammonium sulphate solutions, and therefore ammonium sulphate is generally employed to salt out the impurities and the enzymes in the order desired. After the enzymes are salted out, they can be further purified by recrystallization or other purification methods to produce as products the crystalline enzymes of standard potency and stabilized strength.

The process of this invention is particularly well adapted for producing crystalline pancreatic enzymes, such as crystalline trypsin. It has been found that the presence of an organic solvent in the extract such asmeth anol, ethanol, and acetone interferes with the recovery of the enzymes in crystalline form by ammonium. sulfate fractionation. In fact, if more than 7% of the; organic: solvent is present, it is very difficult to control the crystallization. Preferably the amount or organic solvent: kept below 4%. Optimum results are obtained; when the aqueous extract is substantially free of organic soltrent- Distillation under reduced pressure of the residues themselves, or a slurry formed by the addition of water thereto, permits the organic solvent concentration to be reduced to the point desired, while other methods of reducing concentration are much less effective.

To facilitate the understanding of the process steps described above and the means of carrying out these steps, the accompanying drawing shows in diagrammatic form a simplified flow sheet for removing the enzymes from pancreas residues resulting from the extraction of insulin from pancreas glands. In the upper left-hand corner there is shown a storage tank 10 into which the pancreas residue and the required amount of acidified water are charged. This mixture is pumped from tank 10 into steam still 11 in which the organic solvent is removedby distillation under reduced pressure. The organic solvent, which will generally be ethyl alcohol, is vaporized and leaves through the top of steam still 11 and passes through a condenser 12 in which it is condensed into a liquid and passes into a tank 13. After the removal of the organic solvent, if it is desired to continue the extraction of the pancreas residues, the residues and aqueous solvent can be passedto an extractor 14 which is equipped with an agitator 15. The operation and purpose of extractor 14 is described more fully in co-pending application United States Serial No. 259,090, cited above. After the completion of the treatment of the residues in extractor 14, the mixture is passed to a centrifuge 16 in which the spent residue is separated from the liquid portion containing the enzymes.

The liquid is preferably discharged into a fat skimmer 17, which is a small tank adapted for the removal of any fat or lipoidal matter that is contained in the liquid portion. The fat-free liquid is then passed into an extract tank 18 :in which the ammonium sulphate or other reagent can be added for the separation and activation of the enzymes, as previously described.

Instead of a vacuum still, a vacuum shelf-dryer can be employed to remove the organic solvent. All of the operating conditions previously discussed apply to a shelfdryer as well as to a still. However, it is preferred to employ a still whenever more than 6 parts of water are added to each part of solids in the residues. It will be understood that some water will be removed along with the organic solvent no matter what apparatus is employed 'to distill off the organic solvent. In actual practice it has been found desirable to remove about 2 parts of 'water with every part of organic solvent. Thus, sufiicient water should be present in the residues to prevent the :solids from being reduced to dryness, and there is prefer- .ably an excess of at least 2 parts of water to each part of solids, so that the removing of the organic solvent and the extraction ofa substantial portion of the enzymes will occur simultaneously.

To more fully illustrate the details of this invention, it is desired to set out the following illustrative examples:

Example I The pro-enzymes were recovered from insulin-free beef pancreas residues by the following procedure: The residues contained about 70% liquid to 30% solids by weight, and the liquid portion was 65% ethanol by volume acidified to pH 2.85 with phosphoric acid. 1,000 lbs. of these residues were suspended in 400 gals. of tap water which contained 2200 cc. conc. H2804. been accomplished, utilizing an impeller-type agitator, the slurry was pumped into the still. The distillation was made in a pot-type still, under vacuum, with a median temperature of 60-70 F. and a maximum of F. gallons of liquid, approximately 30% ethanol was removed in this step. The alcohol concentration of the still concentrate was approximately 1%. The concentrate was then diluted with 65 gallons of tap water in order to reach a consistency which'could be circulated.

The diluted liquid was then transported back to its ,original tank and cooled to 40 F. Extraction was accomplished with two 30-minute circulations through a three bladed impeller-type pump. The suspension was zslowly stirred with an impeller-type agitator for 15 min- ,utesbetween and after circulationcycles. This action After suspension had' permitted the fat particles to coalesce before-further extraction and processing. Clotted fat particles were removed during these steps by skimming.

Separation was accomplished in a Bird contrifuge of the cylindrical type. The tissue from the first centrifugation was suspended in 225 gals. of tap water containing 340 cc. of H2804. The washing was accomplished by a 30-minute circulation through the impeller-type pump. The resultant suspension was then centrifuged and the efiluent added to that obtained from the first separation.

0.7 lb. of (NI-102804 was added per gallon of extract. This afforded a .15 saturation with the salt. The salt was added slowly in order to prevent a localized high concentration and a potential irreversible precipitation of protein.

45 minutes of impeller-type agitation was found to be sufiicient for the complete dissolution of the salt.

The separation was accomplished in a Sperry-type press which had been pre-eoated with Hyfio. The pH at this point was found to be critical (1.9-2.1 before salt addition) for there are semi-soluble materials present which. if not precipitated at this pH, not only clog the filter press, but also pass through in the elfiuent and interfere with subsequent processing. The press was washed with 50 gals. of water containing 35 lbs. of (NI-102804, 75 cc. of H2SO4 and lbs. of Hyflo to which had been added the fat removed during the extraction and centrifugation steps. This washing was followed by another 50 gal. portion of the previous washing solution. Air was blown through the press for four hours in order to complete recovery of the filtrate.

4 lbs. of (NH4)2SO4 was added per gallon of filtrate obtained. The solution was then approximately 0.8 sat. with (NH4)2SO4. Dissolution was completely by a 15- minute circulation through the impeller-type pump and precipitation was obtained through a 4-hour agitation with an impeller-type stirrer. This suspension was then filtered through a Sperry-type press, pre-coated with Hifio. Hifio was also added to the slurry; the total amount used being 12 lbs. The filtrate was a proteinfree (NH4)2SO4 solution, which can be recovered. The filter cake was dissolved in 5 cc. of distilled water per gram of cake. The pH was held at 2.8-3.3. 150 g. (NI-102504 was added per liter of solution. The addition was made slowly and the material was agitated vigorously for 15 minutes and allowed to settle for an additional 30 minutes before starting the filtration. The press was washed with liters of a 0.4 saturated (NH4)2SO4 solution. The precipitate, containing chymotrypsinogen B was held at 23 F. for further processing.

200 g. of (NH4)zSO4 was added per liter of filtrate obtained from the last step. The suspension was agitated for minutes and allowed to fiocculate over an additional 30-minute period. The material was then filtered and the precipitate removed to a temperature of 65 F. for further procession. This precipitate contained the crude trypsinogen and chymotrypsinogen.

110 g. of (NH4)2SO4 per liter of solution was added to the filtrate from the last step and the filtered precipitate obtained therefrom was held at 23 F. for further processing. The precipitate was the crude ribonuclease. The precipitate of crude trypsinogen and chymotrypsinogen was dissolved in 1.5 cc. of distilled water per cc. of distilled water per gram of cake and (2) 1.5 cc.

of saturated (NH4)2SO4 solution per gram of cake. After 15 minutes of agitation and 30'rninutes of settling, the

8 suspension was filtered with Hiflo and the precipitate discarded. g. of (NH4)2SO4 was added per liter of filtrate obtained. After the previously described agitation and settling periods, the material was filtered. The filter cake containing the trypsinogen was dried as completely as possible in preparation for crystallization. The trypsinogen cake was cooled to 45 F. by allowing it to stand in a metal pot placed in a freezer vault. Borate "buffer (pH 9.0) was cooled to 60 F. by standing in the cold. The buffer solution was then mixed into the trypsinogen cake very slowly in the ratio of 1.5 cc. per gram of cake with minimal agitation at 32 F. The temperature should never rise about 50 F. This material was allowed to stand 16 hrs. at 32 F. at pH 7.6 One cc. of saturated MgSO4 solution per cc. of buffer-cake solution was added slowly, with continuous agitation. This solution was allowed to stand 3-4 hrs. at 32 F. The solution was seeded with crystalline trypsin and held at 5 C. for 48-72 hrs. for crystallization. The crystalline trypsin was then separated by filtration. Subsequently, the borate buffer salt was removed by dialysis, and after clarification the dialyzed material was packaged in vials.

Example II The procedure of Example I was substantially followed except that pork pancreas residues were substituted for the beef pancreas residues. Because of the greater fat or lipoidal pancreas glands (averaging 30 to 40% as compared to 5 to 10% for beef pancreas glands) special precautions were taken to prevent the fat from interfering with the recovery of the crystalline enzymes. Subsequently to the distillation step, the slurry was refrigerated to around 31 F., and maintained at this temperature during the pump extraction and centrifugation steps to promote clotting and separation of the fat particles.

Another modification in the procedure was the passing of the centrifugate into a small tank immediately after the separation of the spent residue. in this skimmer tank, the centrifugate was held up long enough to allow the fat particles, which had been further coalesced by the centrifugation, to rise to the surface and to be removed by skimming. In this Way, a substantially fatfree extract was obtained.

Example III If it is only desired to recover crystalline trypsin and chymotrypsin, the following can be employed: Insulinfree pancrease residues are suspended in 4 volumes of pH 1.8 water acidified with hydrochloric acid. The pH is then readjusted to 1.8 with hydrochloric acid. The ethanol concentration is then decreased to 3% by distillation under reduced pressure. The enzyme extraction is then completed by passing the slurry into a tank within which it is extracted over a 48 hour period at 37 F. and at apH of 1.8.

The tissue is then separated by centrifugation and discarded. To the centrifugate is added ammonium sulfate until a concentration of 0.4 saturation is reached. The resultant suspension is allowed to settle at 5 C. for 48 hours.

The suspension is then filtered and the precipitate, containing the animal protein fractionation, is discarded. Ammonium sulfate is added to the supernatant to 0.7 saturation, and the resultant suspension is allowed to settle for 48 hours at 5 C. The crude enzyme precipitate is separated, and the supernatant discarded.

The crude 0.7 saturated cake is reworked by dissolving it in 10 volumes of distilled Water and refractionating it through the 0.4 and 0.7 saturated ammonium sulfate steps at 5 C., as described above. The 0.7 saturated cake obtained by these steps can be reworked again by dissolving it in 3 volumes of distilled water and refractionating through the 0.4 and 0.7 ammonium sulfate saturation steps at 25 C.

The 0.7 saturated cake thus obtained is dissolved in 1.5 volumes of distilled waterat 25 C. Saturated ammonium sulfate solution is added to 0.25 saturation. The pH is adjusted to 5.0 with NaOH. The solution is inoculated with a small amount of chymotrypsinogen crystals, and allowed to stand at 25 C. until the chymo trypsinogen crystallization is complete, usually 48 to 72 hours will be required. The suspension is then filtered and the precipitate held for crystalline chymotrypsin preparation. I

The pH of the filtrate subsequent to the separation of the crude chymotrypsinogen is adjusted to 3.0 with 5 N H2804. Ammonium sulfate'is added to this solution to 0.7 saturation. The suspension is filtered'and the supernatant discarded. The precipitate obtained .is dissolved in 3 volumes of distilled water and reworked through a 0.4 and 0.7 ammonium sulfate saturation at 25 C. The 0.7 saturated precipitate is dissolved in 15 volume of pH 9.0 borate buffer at C. The pH of the solution is adjusted to 7.0 with 5 N NaOH. Saturated magnesium sulfate is added to 0.5 saturation. The solution is held at 5 C. until crystallizationof the trypsin is complete, usually 7 to 9 days will be required. The crystallization suspension is separated by filtration and the filtrate discaf'ded. The crystalline trypsin cake may then be held at 5 C. or suspended in 10 volumes of water and lyophalized.

The precipitate of crude crystalline chymotrypsinogen is reworked by suspending it in 3 volumes of distilled water. Saturated ammonium sulfate solution is added to 0.5 saturation. The precipitate obtained is separated and can be again reworked by dissolving it in 3 volumes of distilled water and adding the ammonium sulfate solution to 0.5 saturation. The twice crystallized cake is then suspended in 3 volumes of distilled water and 1 volume of pH 7.5 M. sulfate buffer is then added. The pH of the solution is adjusted to 7.6, and the solution is seated with crystalline trypsin. Activation occurs at 5 C. after a period of 48 hours. The pH of the activated solution is then adjusted to 4.0, and the activated material is precipitated by adding ammonium sulfate to 0.5 saturation. The 0.5 precipitate is separated, and dissolved in volume of acidified water with the pH adjusted to 3.9, and crystallized at 25 C;, usually 24 to 48 hours will be required to complete the crystallization. The crystallized chymotrypsin can then be separated and v recrystallized by dissolving in 1.5 volumes of pH 2.0

water. Saturated ammonium sulfate is added to 0.5 saturation, and the suspension held for a few hours at 25 C. The resulting precipitate of crystalline chymotrypsin is recovered by filtration, the filter cake washed with saturated magnesium sulfate solution at pH 1.3, and held at 5 C.

the proteolytic pro-enzymes. 1,000 lbs. of insulin-free beef pancreas residues were suspended in 425 gallons of water acidified with hydrochloric acid to pH 1.8, and the resulting slurry was readjusted with hydrochloric acid solution to pH 1.8. The total volume of the slurry produced was 520 gallons. 170 gallons of liquid was removed by distillation in vacuo at 70 to 80 R, which effected a substantially complete removal of the ethanol. To the remaining slurry, having a volume of 350 gallons, water was added acidified to pH 8 with hydrochloric acid to dilute the slurry to a culated. The resulting slurry had a volume of 430 gallons. This slurry was passed to a tank in which it was extracted with general agitation at 40 F. for 17 hours.

The slurry was then passed to a Bird-type centrifugation in which the spent residues were separated from the extract. The separated residues were washed with water acidified to pH 1.8 with hydrochloric acid, and the wash water was added to the centrifugate to produce a total consistency which could becirvolume of extract of 515 gallons. The extract was filtered after the addition of 150 lbs. Celite and the cake. was washed with 90 gallons of water to give an extract volume of 550 gallons. The filter cake contained the carboxy peptidase.

The pH of the extract was adjusted to 2.5 concentrated hydrochloric acid and 5 lbs. of ammonium sulfate were added per gallon of extract. suspension was filtered with the aid of Celite to produce a filtrate of 650 gallons of a protein-free ammonium sulfate solution from which the ammonium sulfateiwas recovered. The filter cake was suspended in 3 volumes of distilled water to give a total volume of gallons. 1 grams per liter of ammonium sulfate was then added to the suspension to adjust the concentration to about 0.4 ammonium sulfate saturation. After the formation of the precipitate, the suspension was filtered at 70 F. and the filter cake contained the desoxyribonuclease.

To the filtrate, having a volume of 25 gallons, 165 grams per liter of ammonium sulfate was added to produce a concentration of about 0.65 saturation. After the formation of the precipitate, the suspension was filtered at 70 F. The filtrate contained the ribonuclease, which was recovered by adding 105 grams per liter of ammonium sulfate to about 0.8 saturation, after the formation of the precipitate of ribonuclease was separated by filtration at 70 F. The filtrate was a protein-free ammonium solution from which the ammonium sulfate was recovered.

The 0.65 saturation filter cake contained the trypsinogen and chymotrypsinogen. Crystalline trypsin and chymotrypsinogen were then prepared from this filter cake by the procedure set forth in Example III.

Example V 7 minutes while being generally agitated with an impellertype agitator, and the fat simultaneously removed by skimming. The slurry is then circulated through the impeller-type pump for an additional 30 minutes. The extract is separated from the spent residues by centrifugation, clarified, and a crude trypsinogen precipitate is obtained at 0.8 saturation with ammonium sulfate. The precipitate is then dissolved in 6 volumes of water, and purification is effected by adding a saturated ammonium sulfate solution at a concentration between 0.35 to 0.55 I saturation. The trypsin is then crystallized at pH 7.1.

to 7.5 in a borate buifer solution at 0.3 saturation with magnesium sulfate.

dialyzed material is packaged in vials.

Example VI In the foregoing examples, the trypsin is obtained in crystalline form. Sometimes additional purifying steps beyond those described will be desirable to prepare a pure crystalline product of stabilized potency and standardized strength. With particular reference to the procedure described in'Example I, occasionally after the trypsinogen cake has been dissolved in the borate buffer solution, and the magnesium sulfate solution added, noticeable gelatinization will appear in the standing solution, or the solution may be of dark color or dingy appearance. If any of these conditions occur, it is desirable to subject the solution to filtration before proceeding with the crystallization of the trypsin. Also, subsequent to the crystallization the crystalline product may appear gummy or gelatinous. When this occurs the separated crystalline The resulting The slurry- The borate salt is removed from the, crystalline trypsin by dialysis, and after clarification the.

solution should be centrifuged using a batch-bowl. The precipitate from the centrifugation should then be suspended in a minimal amount of pH 9.0 borate buffer solution. This suspension should then be filtered and dried as much as possible. The dry filter cake thus obtained can be stored at 23 F. indefinitely.

While in the foregoing specification this invention has been described in considerable detail for purpose of illustrating embodiments thereof, it will be apparent that many of these details can be varied widely without departing from the spirit of the invention.

We claim:

1. In a process for recovering enzymes of the group consisting of ribonuclease, chymotrypsinogen B, alpha chymotrypsinogen and trypsinogen from the solvent-rich residues resulting from the extraction of insulin from comminuted pancreas glands by contacting said glands with an acidified, water miscible organic solvent for insulin, the steps of adding up to 20 parts by weight of water to each part by weight of solids in said residues, and then decreasing the concentration of the organic solvent in said mixture to less than 20% by volume by subjectingsaid mixture to distillation under reduced pressure at a temperature of from 60 to 100 F. for a period of over 4 hours while maintaining the pH of said mixture below pH 4, whereby the major portion of the proteolytic enzymes in said residue can be extracted in a relatively short period of time without destroying any substantial amount of said enzymes.

2. In a process for recovering enzymes of the group consisting of ribonuclease, chymotrypsinogen B, alpha chymotrypsinogen and trypsinogen from the solvent-rich residues resulting from the extraction of insulin from comminuted pancreas glands by contacting said glands with an acidified organic solvent for insulin selected from the group consisting of methanol, ethanol, and acetone, the steps of adding from 6 to 14 parts by weight of water to each part by weight of the solids in said residues and a sufiieient quantity of a strong inorganic acid to reduce the pH of the resulting mixture to below pH 2.5, and then decreasing the concentration of the organic solvent to less than 4% by volume by subjecting said mixture to distillation under reduced pressure for from 4 to 10 hours at a temperature of from 60 to 100 F., whereby the major portion of the proteolytic enzymes in said residue can be extracted in a relatively short period of time without destroying any substantial amount of said enzymes.

3. In a process for recovering enzymes of the group consisting of ribonuclease, chymotrypsinogen B, alpha chymotrypsinogen and trypsinogen from the solvent-rich residues resulting from the extraction of insulin from comminuted pancreas glands by contacting the solids in said glands with a solvent for insulin consisting of an acidified ethanol and water mixture containing at least 50% ethanol by volume, the steps of adding from 6 to 14 parts by Weight of water to each part by weight of solids in said residues and a sufficient quantity of a strong inorganic acid to reduce the pH of the resulting mixture to below pH 2.5, and then decreasing the concentration of the organic solvent in said mixture to less than 4% by volume by subjecting said mixture to distillation under reduced pressure for over 4 hours at a temperature of from 60 to 100 F., whereby the major portion of the proteolytic enzymes in said residue can be extracted in a relatively short period of time without destroying any substantial amount of said enzymes.

4. In a process for recovering enzymes of the group consisting of ribonuclease, chymotrypsinogen B, alpha chymotrypsinogen and trypsinogen from the solvent-rich residues resulting from the extraction of insulin from comminuted pancreas glands by contacting said glands with an acidified, water-miscible organic solvent for insulin, the steps of adding from 6 to 14 parts by weight of water to each part by weight of the solids in said residues together with a sufficient quantity of a strong inorganic acid selected from the group consisting of sulphuric acid and hydrochloric acid to reduce the pH of the resulting mixture to below pH 2.5, and then decreasing the concentration of the organic solvent in said mixture to less than 4% by volume by subjecting said mixture to distillation under reduced pressure for at least 4 hours at a temperature from 60 to F., whereby the major portion of the proteolytic enzymes in said residue can be extracted in a relatively short period of time without destroying any substantial amount of said enzymes.

5. In a process for recovering enzymes of the group consisting of ribonuclease, chymotrypsinogen B, alpha chymotrypsinogen and trypsinogen from the solvent-rich residues resulting from the extraction of insulin from comminuted pancreas glands by contacting said glands with an acidified, water-miscible organic solvent for insulin, the steps of adding from 6 to 14 parts by weight of water to each part by weight of solids in said residues, and then decreasing the concentration of the organic solvent in said mixture to less than 20% by volume by subjecting said mixture to distillation under reduced pressure at a temperature between 60 and 100 F. for a period of over four hours while maintaining the pH of said mixture below pH 4, whereby the major portion of the enzymes of the group consisting of ribonuclease, chymotrypsinogen B, alpha chymotrypsinogen and trypsinogen in said residue can be extracted in a relatively short period of time without destroying any substantial amount of said enzymes.

6. In a process for recovering enzymes of the group consisting of ribonuclease, chymotrypsinogen B, alpha chymotrypsinogen and trypsinogen from the solvent-rich residues resulting from the extraction of insulin from comminuted pancreas glands by contacting the solids in said glands with a solvent for insulin consisting of an acidified ethanol and water mixture containing at least 50% ethanol by volume, the steps or" adding from 6 to 14 parts by weight of water to each part by weight of solids in said residues and a sufficient quantity of a strong inorganic acid to reduce the pH of the resulting mixture to below pH 4.0, and then decreasing the concentration of the organic solvent in said mixture to less than 20% by volume by subjecting said mixture to distillation under reduced pressure for a period of from 4 to 10 hours at a temperature ranging from 60 to 100 F.

References Cited in the file of this patent UNITED STATES PATENTS 2,503,313 Levin Apr. 11, 1950 2,529,152 Grant Nov. 7, 1950 2,571,126 Frederickson Oct. 16, 1.951

2,637,680 Petersen May 5, 1953 FOREIGN PATENTS 618,174 Great Britain of 1946 OTHER REFERENCES Tauber: Chem. and Tech. of Enzymes, John Wiley and Sons, Inc., New York (1949) page 148. 

1. IN A PROCESS FOR RECOVERING ENZYMES OF THE GROUP CONSISTING OF RIBONUCLEASE, CHYMOTRYPSINOGEN B, ALPHA CHYMOTRYPSINOGEN AND TRYPSINOGEN FROM THE SOLVENT-RICH RESIDUES RESULTING FROM THE EXTRATION OF INSULIN FROM COMMINUTED PANCREAS GLANDS BY CONTACTING SAID GLANDS WITH AN ACIDIFIED, WATER MISCIBLE ORGANIC SOLVENT FOR INSULIN, THE STEPS OF ADDING UP TO 20 PARTS BY WEIGHT OF WATER TO EACH PART BY WEIGHT OF SOLIDS IN SAID RESIDUES, AND THEN DECREASING THE CONCENTRATION OF THE ORGANIC SOLVENT IN SAID MIXTURE TO LESS THAN 20% BY VOLUME BY SUBJECTING SAID MIXTURE TO DISTILLATION UNDER REDUCED PRESSURE AT A TEMPERATURE OF FROM 60* TO 100* F. FOR A PERIOD OF OVER 4 HOURS WHILE MAINTAINING THE PH OF SAID MIXTURE BELOW PH4, WHEREBY THE MAJOR PORTION OF THE PROTEOLYTIC ENZYMENS IN SAID RESIDUE CAN BE EXTRACTED IN A RELATIVELY SHORT PERIOD OF TIME WITHOUT DESTROYING ANY SUBSTANTIAL AMOUNT OF SAID ENZYMES. 